The efficacy of enhanced aqueous extracts of Melia azedarach leaves and fruits integrated with the Camptotylus reuteri

Bulletin of Insectology 56 (2): 269-275, 2003 ISSN 1721-8861 The efficacy of enhanced aqueous extracts of Melia azedarach leaves and fruits integrated with the Camptotylus reuteri releases against the sweetpotato whitefly nymphs Chantal JAZZAR 1, Efat ABOU-FAKHR HAMMAD 2 1 Institute of Plant Protection and Plant Diseases, Hannover University, Germany 2 Faculty of Agricultural and Food Sciences, American University of Beirut, Lebanon Abstract Bioassays with aqueous extracts of different growth stages of Melia azedarach L. (chinaberry) leaves and fruits were conducted against 3 rd - 4 th instar nymphs of Bemisia tabaci (Gennadius) on tomato plants. Mortality of instars was assessed 15 days posttreatment. Results illustrated that every Melia extract produced a significant greater mortality of B. tabaci than the controls. Extracts combined with the surfactant were significantly more toxic than those applied alone. Extracts with Tween-20 caused a mortality range of 34.6 to 67.9% and 53.5 to 74.1% for leaves and fruits, respectively. These was parallel to 15.6 and 31.4% and 11.4 to 18.7% for leaf and fruit extracts, lacking the surfactant, respectively. The synchronous application of one indigenous adult mirid predator, Camptotylus reuteri (Jakovlev) introduced at 30 min after spraying the Melia fruit extract, for a period of 3 days, was not synergistic with the botanical extract. The potential insecticidal activity of Melia extracts appears to be enhanced by Tween-20, and they are safe to integrate with C. reuteri biocontrol agent as the extracts did not cause any mortality of the predator. Key words: Melia azedarach, Bemisia tabaci, Camptotylus reuteri, Tween-20, botanical insectici. Introduction Bemisia tabaci (Gennadius) (Homoptera Aleyrodidae), variously known as the cotton, tobacco or sweetpotato whitefly, is a highly polyphagous pest record from more than 500 host plant species (Greathead, 1986) representing more than 60 plant families (Mound and Halsey, 1978). All stages are difficult to control with conventional insecticis because of their high reproductive rate and their preferred habitat on the unrsurface of leaves (Cahill et al., 1996). B. tabaci nymphs and adults cause direct damage by piercing and sucking cell contents and in excreting huge amounts of honeyw that promotes sooty mould fungal velopment and reduces the photosynthetic efficiency of the plant and thus yield. Indirect damage is manifested by the transmission of more than 50 geminiviruses including the tomato yellow leaf curl virus (TYLCV) attacking tomatoes grown commercially worldwi (Markham et al., 1996). B. tabaci is notorious for its resistance to an array of pesticis extending from the pyrethroids, organophosphates (OPs) and carbamates (Cahill et al., 1994) to the insect growth regulators buprofenzin and pyriproxifen (Horowitz et al., 1994). Hence, consirable effort has been ma towards the discovery and velopment of alternatives for management of this insect with reduced hazards to the environment and human health. Botanicals offer a potentially safe, environmentally sound and effective tool to suppress some threatening pests. Among botanicals, preparations from the neem tree Azadirachta indica A. Juss (Meliaceae), with its potent active ingredient, azadirachtin, have been thoroughly studied as crop protectants. Azadirachtin is a tetranortriterpenoid with behavioural and physiological bioactivities in more than 200 species of insects, extremely low mammalian toxicity, some selectivity favoring biocontrol agents and systemic action in a variety of important crop plants (Isman, 1997). Botanicals such as neem or chinaberry contain a mixture of active constituents, which might diffuse the selection process, mitigating the velopment of resistance compared to that expected with a single active ingredient (Feng and Isman, 1995). Provid it is compatible with biocontrol agents, the Persian lilac or Chinaberry tree, Melia azedarach L. (Meliaceae), a close relative of neem, presents a promising approach for integrated pest management. Depending on the source material and the pest in question, the insecticidal potency of M. azedarach extracts could be equivalent to that of neem extracts (Champagne et al., 1989; Lee et al., 1991). M. azedarach is abundant in Lebanon and other Mediterranean countries where it is mainly planted for sha. The main objective of our study was to explore the utilization of a local plant resource for the management of B. tabaci on tomato by testing aqueous extracts of M. azedarach leaves and fruits on the 3 rd - 4 th instars of B. tabaci. We also examined the effect of integrating a local mirid predator Camptotylus reuteri (Jakovlev) (Heteroptera Miridae) with M. azedarach fruit extracts. Materials and Methods Insect Material A laboratory colony B. tabaci biotype A, more than five years old, originally collected from field populations on beans and cucumbers grown in coastal areas of Lebanon was reared on broccoli Brassica oleraceae L. cv. Calabrese Natalino and eggplants Solanum melon-

gena L. cv. Blackbeauty. Whiteflies were maintained in a cage (140 85 130 cm) entirely covered with fine mesh screen (270 770 µm) in a glasshouse at 25 ± 2 C, 80 ± 10 % RH, and a 16:8 L:D photophase, located on the campus of the American University of Beirut (AUB), Beirut, Lebanon. A continuous supply of new plants was provid as need for the colony replenishment. The colony was supplemented annually with leaves highly infested with 3 rd - 4 th instars of B. tabaci collected from the field; only emerging adults from the latter were introduced because of the menace of contamination with other intrur parasitoids and arthropods if nymph infested leaves were to be supplied to the colony. Adults and nymphs of C. reuteri were collected from a field of eggplants infested with B. tabaci, at Choueifat (in the middle coastal area of Lebanon) during September-October, 1998. The mirids were removed by a manual aspirator, transferred in a cooler from the field to the glasshouse compartment and released in an insect proof cage (74 145 95 cm). The cage was placed in a glasshouse at an average temperature of 27 ± 3 C, 65 ± 20% RH and continuous light. C. reuteri were exposed to the above conditions for four days before experimental use. They were maintained on whitefly-infested eggplants and Myzus persicae (Sulzer) (Homoptera Aphididae) collected from the AUB campus and introduced into the colony. Sponges soaked in distilled water were placed in the colony to provi water for the predator. Plant Material Tomato plant preparation Tomato seeds (Lycopersicon esculentum L., cv. Ronda 44) were planted in trays containing a soil mixture of peatmoss:perlite (3:1). Seedlings with the first true leaves were drenched with fungicis Rhizolex (Tolclofos-methyl) and Tachigaren (Hymexazole) to avoid damping-off disease and then transplanted into 12 cm plastic pots containing the same potting mixture. Each seedling was placed in a cylindrical insect proof plastic cage (30 cm high by 20 cm diameter) covered with mesh (270 770 µm) firmly held with a plastic lid at the top. Aeration in the cage was enhanced by drilling four holes each of 7.5 cm diameter in the body of the cage that were covered with mesh. An opening of 2.5 cm in diameter was also drilled at the level of the soil in the pot for watering during the experimental period. Ten adult whiteflies were collected from the rearing colony by a manual respirator and then introduced in a glass vial into each cage. Two days later, plants were checked for oviposition. Two weeks later, forty 3 rd - 4 th instars of B. tabaci were counted at random per plant and were marked on one or more tomato leaves. Preparation of M. azedarach extracts Source of plant material Leaves and fruits of M. azedarach trees located on the AUB campus were collected during June 1996 (leaves and green fruits) and November 1996 (mature fruits). The leaves were classified into different age classes according to their maturity: old, from the lowest part of the whole branch, medium, from the middle part, and young, from the tip of the main branch. Each plant part was weighed and stored in a plastic bag at -20 C. When need, the frozen plant material was removed from the freezer, soaked for 15 minutes in distilled water then dried at ambient laboratory temperature for 6-12 hours. Extraction of plant material Lots of 100g dry weight of each sample of leaves or fruits and 70g of mature fruits only for C. reuteri bioassay, were ground with distilled water in a blenr (16,000 rpm; Sorvall Omnimixer, USA) at a ratio of 1:5 (w/v), and soaked for 48 hours at room temperature. The extracts were filtered through cheesecloth, followed by vacuum filtration through filter paper (Whatman no. 40). Tween-20 was add as an adjuvant to the Melia extracts at a rate of 0.5%. The extracts were applied to the whitefly-infested plants till runoff using SPRA- Tool (Crown, Illinois, USA) sprayers. Experimental Set-up All bioassays were performed in the glasshouse at 25 ± 2 C, RH of 80 ± 10%, and 16:8 L:D photoperiod. Each plant received an average of 18.2 ml of the Melia extract. The treatments includ 6 extracts of each of the leaf and fruit material with and without Tween-20 (0.5%), distilled water plus Tween-20 (0.5%) and distilled water alone as controls. Mortality was assessed by counting insects with melanotic spots in the late third instars or leaky symptom/oozing in the late fourth instars or incomplete emergence of adults from the nymphal exuviae. Five treatments with the introduction or absence of the predator includ: mature fruit extracts with and without Tween 20 (0.5%), Melia extract with adjuvant in the presence of the predator, distilled water with adjuvant in presence of the predator and an unsprayed plant to which C. reuteri was introduced. Each plant in treatments including Melia extracts received an average of 25 ml of the solution. The mirid was introduced on the sprayed plant 30 minutes after spraying. The experimental duration was 72 hours. Each treatment was replicated three times and the entire experiment was repeated twice. The observed feeding symptom of the adult C. reuteri on B. tabaci instars was in the form of a small hole in the body of each instar. Statistical analysis The statistical package MSTAT-C (Anonymous, 1991) was used for data analysis. For the bioassays with various M. azedarach aqueous extracts, the data was collected in the form of percent mortality of the 3 rd - 4 th instars of B. tabaci, corrected with respect to the distilled water control based on Abbott s (1925) formula, then transformed into arcsin x, x being the percent mortality of nymphs, to normalize the mortality. The sign was a completely randomized sign (CRD) and the analysis of variance was performed over the factor Melia age and presence or absence of Tween-20. The data assembled from the integration of mature Melia fruit extracts and the adult predator on the 3 rd - 4 th instars of B. tabaci was transformed into arcsin x, x be- 270

ing the percent mortality to normalize the data. The experiment was laid out as a completely randomized block sign (RCBD) with multiple observations. All means were separated by Fisher s (1960) LSD test if significant F values were obtained (Gomez and Gomez, 1984). Further analysis for the association between the Melia extract and the adjuvant Tween-20, and the additive and non-additive interactions between the predator and the adjuvant were termined separately using a modified procedure by McVay et al. (1977) and Salama et al. (1984) for probit analysis. The expected additive proportional mortality (Me) of the Melia extract-tween-20 combinations was calculated by: Me = Mn + Mi (1 - Mn), where Mn and Mi are the observed proportional mortalities caused by the Melia extract and Tween-20 alone, respectively. Results from the chi-square test, χ 2 = (Mni - Me) 2 / Me, where Mni is the observed mortality due to Melia extract and Tween-20 combinations, were compared to a chi-square table for 1 df, α = 0.05. If the calculated chi-square value exceed the table value, a non-additive effect (i.e. synergistic or antagonistic) was assumed (Finney, 1964). Furthermore, if the difference Mni - Me = D had a positive value, a significant interaction was consired synergistic; if D had a negative value, a significant interaction was consired antagonistic. Results and discussion Bioassay of the M. azedarach aqueous extracts on the 3 rd - 4 th instar nymphs of B. tabaci indicated that there were significant differences in percent mortality among treatments (F = 5.25; 11, 24; P<0.05). The percent mortality caused by the extracts with Tween-20 ranged from 34.6 to 67.9% for leaves, and from 53.5 to 74.1% for fruits. Mortality caused by the extracts alone (without Tween-20), ranged from 15.6 to 31.4% for leaves and from 11.4 to 18.7% for fruits (figure 1). There were no significant differences in percent mortality of instars among leaf or fruit extracts of different maturity periods in the absence of Tween-20. These extracts were comparable in their effects on B. tabaci nymphs to distilled water plus Tween-20 (figure 1). All plant extracts with Tween-20 were significantly more effective than the distilled water control (figure 1). The high activity of these extracts against B. tabaci instars was verified by the termined synergistic effect of Tween-20 with each extract (table 1). The enhancement of Melia activity by Tween-20 can be explained by the fact that Tween-20 likely facilitates the absorption of the active principles in M. azedarach through the waxy cuticle of the whitefly nymphs, and/or facilitates the movement of the active principles into the parenchymal cells of the host plant, from which they are ingested by nymphal whiteflies. In this study, three symptoms due to Melia toxins were observed on the whitefly instars and were used to assess mortality. The first symptom, on late fourth instars, was exudation of body fluids through tissue disruption or oozing symptom noted three to four days after treatment. The second symptom was manifested by adults that could not emerge from the pupal exuvia, represented by 2.5-7.5% of ad instars. The last symptom was 3 rd instars that turned brownish due to dried insect body. Schlüter (1995) proposed the cellular mechanism that unrlies the oozing or leaky symptom caused by azadirachtin 1-2 days after application as fat cells starting to separate from intact neighbour cells. He further attributed the dry brown pupa symptom observed in the third instar to melanization, a fense reaction leading to massive tissue generation (%) Mortality of instars 100 80 60 40 20 0 a ab bcd a abc bcd c e LO-T LO LM-T LM LY-T LY GF-T GF MF-T MF DW-T DW Treatments Figure 1. Effect of semiqualitative age classes of M. azedarach aqueous extracts on 3 rd - 4 th instars of B. tabaci. Means were separated by LSD test (P < 0.05). Columns head by the same letters are not significantly different. Treatments includ: LO-T = Leaf old + Tween; LO = Leaf old; LM-T =Leaf medium + Tween; LM = Leaf medium; LY-T = Leaf young + Tween; LY =Leaf young; GF-T = Green fruits + Tween-20; GF = Green fruits; MF-T = Mature fruits + Tween; MF = Mature fruits; DW-T = Distilled water + Tween; DW = Distilled water; T = Tween-20 add at 0.5% to some extracts. 271

Table 1. Determination of the synergistic effect* between semiqualitative age classes of M. azedarach leaves and fruits and Tween-20 (0.5%) against B. tabaci **. Melia extracts Proportion Mortality Mn Mi Mni Me χ 2 calculated χ 2 tabulated D Old leaves 0.1887 0.1633 0.6661 0.3212 0.3704 0.0039 0.3449 Medium leaves 0.1121 0.1633 0.5956 0.2571 0.4457 0.0039 0.3385 Young leaves 0.2401 0.1633 0.7408 0.3642 0.3849 0.0039 0.3766 Green fruits 0.0648 0.1633 0.7257 0.2175 1.1873 0.0039 0.5082 Mature fruits 0.1358 0.1633 0.4860 0.2792 0.1526 0.0039 0.2064 *Finney s (1964) method modified by McVay et al. (1977) and Salama et al. (1984) **All mortality means were adjusted with respect to the control, distilled water (5.6% mortality) according to Abbott s (1925) Mn = Observed proportional mortality caused by each Melia extract alone Mi = Observed proportional mortality caused by Tween-20 (0.5%) Me = Expected proportional mortality for the Melia-Tween-20 combination Mni = Observed proportional mortality due to the Melia-Tween-20 combination χ 2 calculated = (Mni-Me) 2 /Me χ 2 tabulated (df =1, P = 0.05) D = Mni-Me (D is positive implies synergism; D is negative implies antagonism) and loss of body contents ensuing in drying. Ascher et al. (1995) related the half emerging pupae symptom to the presence of meliacin and meliacarpin compounds that were tected in the methanolic extracts of green chinaberry fruits, whereas Isman (1997) explained this symptom by disturbance of hormonal balance as release of ecdysteroid causing ecdysal failure when the adult emerges from the nymphal exuvia. The similarity in symptoms between M. azedarach and neem can be related to the analogy of some Melia toxins to azadirachtin that were isolated from fruits of M. azedarach (Kraus et al., 1987). The ecdysis-inhibiting properties of neem might be present in M. azedarach compounds as suggested by both Valladares et al. (1997) and Cabral et al. (1996). This fluctuation in chemistry of M. azedarach leaves and fruits (ripe and unripe) was also noted by Valladares et al. (1999) testing the repellent and insecticidal properties against eggs and nymphs of Triatoma infestans Klug (Heteroptera Reduviidae), the unripe fruit being the most effective. % Mortality of instars 80 70 60 50 40 30 20 10 0 70.4 a 63.4 ab 41.1 bc 39.8 c 16.0 cd 0.8 d Melia MFF* with Tween-20 + mirid Melia MFF + Tween-20 Water + Tween-20 Water + Tween-20 + mirid Melia MFF Untreated plant + mirid Treatments Figure 2. Effect of M. azedarach fruit extract and the indigenous mirid C. reuteri on the 3 rd - 4 th instars of B. tabaci. Means were separated by LSD (P<0.05) test. Columns head by the same letters are not significantly different. * MFF = Mature fruit frozen. 272

Table 2. Determination of the synergistic effect between Melia fruit extract and Tween-20 (0.5%) against B. tabaci. Proportion Mortality χ Mni Mi Mn Me 2 calculated χ 2 tabulated D 0.6343 0.4109 0.1597 0.5050 0.0331 0.0039 1.293 *Finney s (1964) method modified by McVay et al. (1977) and Salama et al. (1984) **All mortality means were adjusted with respect to the control, distilled water (5.6% mortality) according to Abbott s (1925) Mn = Observed proportional mortality caused by each Melia extract alone Mi = Observed proportional mortality caused by Tween-20 (0.5%) Me = Expected proportional mortality for the Melia-Tween-20 combination Mni = Observed proportional mortality due to the Melia-Tween-20 combination χ 2 calculated = (Mni-Me) 2 /Me χ 2 tabulated (df =1, P = 0.05) D = Mni-Me (D is positive implies synergism; D is negative implies antagonism) Mature fruit extracts of M. azedarach combined with the mirid species on the 3 rd - 4 th instar nymphs of B. tabaci led to significant differences (F = 9.76; df = 5, 23; P < 0.05) in nymphal mortality among treatments. However, there was no significant difference in mortality of nymphs between the integrated treatment, i.e. Melia extract with Tween-20 and the mirid predator C. reuteri, and treatment with the extract and Tween-20 alone (figure 2). In no case did C. reuteri enhance nymphal mortality in conjunction with an extract or Tween-20 alone, nor was the mirid effective by itself (figure 2). This result verifies that Tween-20 can have a higher trimental effect on the instars compared to the mirid effect alone (0.83%) unr the tested experimental conditions. The percent mortality due to the mirid feeding in the combined treatment was 5.7% compared to 31.6% due to Tween-20. Our observations showed that the ad instars due to Melia treatment were not fed upon by the mirid and the Melia treatments were not trimental to the predator but might have repelled it from feeding. The insect was often found on new foliage or partially treated foliage as a mean to sustain itself. Our results agree with those of Valladares et al. (1997) who found that both larvae and adults of Xanthogalleruca luteola (Muller) (Coleoptera Chrysomelidae) were terred from feeding on leaves treated with ethanolic extracts of M. azedarach mature fruits. They noted that the antifeedant effect was inpennt from the concentration and was apparent even at the lowest concentration tested. Antifeedant effects of M. azedarach extracts are known for some other insects (Chiu, 1984; Saxena et al., 1984; Ascher, 1987; Kraus et al., 1987; Lee et al., 1991). C. reuteri behaviour could be explained by the fact that host selection is governed by the responses of the insect s gustatory and olfactory sensilla to some compounds acting on feeding behaviour (Blaney and Simmonds, 1996; Nicol et al., 1995). This is also supported by the avoidance of paper refuge treated with M. azedarach extracts shown by T. infestans (Palacios et al., 1993). Feeding terrence without previous contact with the compound has also been reported in experiments with neem (Saxena, 1989). Similarly, significant difference in the mortality of instars between Melia extracts with Tween-20 and Melia extracts alone was tected in the experiment with the mirid. This could be related to the oily nature of Tween- 20, which either promotes the penetration of aqueous Melia extracts through the storage protein and nse fat bodies of the instars or facilitates the absorption of hydrophilic active constituents of the Melia fruit extracts through the waxy surface of the epirmal layer of the leaf into the parenchyma tissue from which the insects take it up during feeding. This is suggested by the synergy tected between the Melia fruit extracts and the emulsifier Tween-20 (table 2). Also, it is unlikely that adults emerging from Melia-treated pupae would be able to produce the same damage as normal ones, for example in terms of oviposition rate (Abou-Fakhr Hammad et al., 2001). According to Shmutterer (1990), the fecundity of homopterous insects is strongly influenced by neem extracts or azadirachtin. Coudriet et al. (1985) found that B. tabaci confined to cotton treated with neem seed extracts posited > 80 % fewer eggs compared with controls up to 7 d after treatments. Also Lowery and Isman (1996) found that exposure to azadirachtin reduced the fertility (live offspring per aphid per day) and the fecundity (live + ad offspring per aphid per day) of adults green peach aphid M. persicae, lettuce aphid Nasonovia ribisnigri (Mosley), and strawberry aphid Chaetosiphon fragaefolii (Cockerell), in a linear concentration-pennt manner. Thus the insecticidal effect of Melia should not be unrestimated by consiring emerging adults as having escaped control. Valladares et al. (1999) noted that the nymphs of T. infestans reared in refuge treated with ethanolic extracts of different age classes of leaves and fruits of M. azedarach were noticeably smaller and lighter in color than respective controls after exposure to the extract for one instar, suggesting that the overall effects could be dramatic if accumulated through the insect s life cycle. Conclusions We have monstrated that extracts of Melia leaves were as effective as those of fruits in causing mortality of B. tabaci 3 rd - 4 th instars. In addition, efficacies of both the leaf and fruit extracts are significantly enhanced by the emulsifier Tween-20, verified by the synergistic effect. Owing to the gradation of the active principles in these extracts, repeated applications may be required for effective suppression of B. tabaci popu- 273

lations. The efficacy of the Melia extract could be enhanced by using a more proficient extraction procedure, because the quantity of the biologically active compounds varies with the extraction method (Flint and Parks, 1989). The lipophilic portion of the Melia extract i.e., oil, may be a valid approach, especially against insectici resistant pests. The limited impact of C. reuteri in our study may be attributed to intrinsic factors related to the predator or to the fact that the insect was exposed to infested tomato plants in its rearing colony for only a few days. A conditioning period might be necessary to enhance predator activity unr the experimental conditions. Furthermore, the aquate timing of the synchronous implementation of a botanical insectici enjoying a repellent activity such as neem or Melia with a natural enemy is of primordial importance lineating the impact of the natural enemy in a sound pest management tactic. Acknowledgments I acknowledge with gratitu F. 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